Abstract
Transgenic mouse models can be subdivided into two main categories based on genomic location: (1) targeted genomic manipulation and (2) random integration into the genome. Despite the potential confounding insertional mutagenesis and host locus-dependent expression, random integration transgenics allowed for rapid in vivo assessment of gene/protein function. Since precise genomic manipulation required the time-consuming prerequisite of first generating genetically modified embryonic stem cells, the rapid nature of generating random integration transgenes remained a strong benefit outweighing various disadvantages. The advent of targetable nucleases, such as CRISPR/Cas9, has eliminated the prerequisite of first generating genetically modified embryonic stem cells for some types of targeted genomic mutations. This chapter outlines the generation of mouse models with targeted genomic manipulation using the CRISPR/Cas9 system directly into single cell mouse embryos.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gordon JW, Scangos GA, Plotkin DJ et al (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A 77(12):7380–7384
Palmiter RD, Brinster RL, Hammer RE et al (1982) Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300(5893):611–615
Yan B, Li D, Gou K (2010) Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line. BMC Mol Biol 11:58. https://doi.org/10.1186/1471-2199-11-58
Dai J, Cui X, Zhu Z et al (2010) Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos. Int J Biol Sci 6(7):756–768
Yan BW, Zhao YF, Cao WG et al (2013) Mechanism of random integration of foreign DNA in transgenic mice. Transgenic Res 22(5):983–992. https://doi.org/10.1007/s11248-013-9701-z
Doetschman T, Gregg RG, Maeda N et al (1987) Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330(6148):576–578. https://doi.org/10.1038/330576a0
Carbery ID, Ji D, Harrington A et al (2010) Targeted genome modification in mice using zinc-finger nucleases. Genetics 186(2):451–459. https://doi.org/10.1534/genetics.110.117002
Yang H, Wang H, Shivalila CS et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379. https://doi.org/10.1016/j.cell.2013.08.022
Sung YH, Kim JM, Kim HT et al (2014) Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24(1):125–131. https://doi.org/10.1101/gr.163394.113
Fujii W, Kawasaki K, Sugiura K et al (2013) Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res 41(20):e187. https://doi.org/10.1093/nar/gkt772
Hill JT, Demarest BL, Bisgrove BW et al (2014) Poly peak parser: method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products. Dev Dyn 243(12):1632–1636. https://doi.org/10.1002/dvdy.24183
Dmitriev DA, Rakitov RA (2008) Decoding of superimposed traces produced by direct sequencing of heterozygous indels. PLoS Comput Biol 4(7):e1000113. https://doi.org/10.1371/journal.pcbi.1000113
Quadros RM, Miura H, Harms DW et al (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18(1):92. https://doi.org/10.1186/s13059-017-1220-4
Yoshimi K, Kunihiro Y, Kaneko T et al (2016) ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun 7:10431. https://doi.org/10.1038/ncomms10431
Luo C, Zuniga J, Edison E et al (2011) Superovulation strategies for 6 commonly used mouse strains. J Am Assoc Lab Anim Sci 50(4):471–478
Spearow JL, Barkley M (1999) Genetic control of hormone-induced ovulation rate in mice. Biol Reprod 61(4):851–856
Hasegawa A, Mochida K, Inoue H et al (2016) High-yield superovulation in adult mice by anti-inhibin serum treatment combined with estrous cycle synchronization. Biol Reprod 94(1):21. https://doi.org/10.1095/biolreprod.115.134023
Morbeck DE, Leonard PH (2012) Culture systems: mineral oil overlay. Methods Mol Biol 912:325–331. https://doi.org/10.1007/978-1-61779-971-6_18
Bin Ali R, van der Ahe F, Braumuller TM et al (2014) Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer. Transgenic Res 23(4):691–695. https://doi.org/10.1007/s11248-014-9802-3
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Scott, G.J., Gruzdev, A. (2019). Genome Editing in Mouse Embryos with CRISPR/Cas9. In: Allen, I. (eds) Mouse Models of Innate Immunity. Methods in Molecular Biology, vol 1960. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9167-9_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9167-9_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9166-2
Online ISBN: 978-1-4939-9167-9
eBook Packages: Springer Protocols